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Bacterial Bioluminescence
What is bio-luminescence? Bioluminescence is the production and emission of light from an organism. Many of these luminescent organisms are microbes (such as Vibrio fischeri) that live in marine environments. They are either free living or can colonize different higher order animals such as the bobtail squid. These animals "light up" for various reasons like attracting prey, attracting a mate, or for camouflage. How does bioluminescense work? The reaction for light production is catalyzed by the enzyme luciferase. The enzyme is a mixed function oxidase consisting of two subunits- alpha and beta. To generate light, luciferase catalyzes the oxidation of a reduced flavin and a long chain aldehyde, producing oxidized flavin and the corresponding long chain fatty acid (Engebrecht, 773). These bacteria do not glow all the time and because of that, this is a highly regulated process. It is turned "on" and "off" by molecules called autoinducers. These molecules are produced by these luminous bacteria. Usually, autoinducers are maintained at a basal or low level in the cell. When the concentration of these molecules increases, it hits a certain threshold. At this point, the autoinducer binds to a regulator protein and starts to transcribe the DNA needed for both parts of the luciferase enzyme. There are two genes that encode for the lucifierase enzyme - Lux A and Lux B. Bacterial Bio-luminescence: Isolation and Genetic Analysis of Functions from Vibrio fischeri In this study, Engebrecht et. al wanted to understand how this system is regulated in V. fischeri. Their experiment consisted of four major steps: Isolation and transformation of LuxA and LuxB, Mutagenesis, and Determination of bioluminescence. Isolation and Transformation of LuxA and LuxB: DNA was isolated from V. fischeri strain MJ-1 and BamHI cut sites were used on this DNA to ligate into their plasmids. After the DNA was isolated, they needed to transform these genes into E.coli cells. They used E.coli ''as their model organism because it is used a lot in molecular cloning experiments, especially transformations. Their genome has been completely sequenced and they grow fairly quickly. Additionally, a lot of researchers work with E.coli and a lot is known about this microorganism. After the transformation, they needed to test and make sure that these colonies could produce light. They tested almost 10,000 colonies and found three colonies that were fully able to produce light. Some of these colonies needed additional amounts of aldehyde added to them because the light that was produced was not bright enough. The three colonies that were ''E.coli ''recombinants were pJE202, pJE201, and pJE205. The only recombinant colony that was from ''V. fishceri ''was MJ-1. Recombinant pJE201 and 202 appeared to encode the enzymatic functions and regulatory proteins the best and were used in the subsequent experiments. '''Mutagenesis:' The next step was to perform an insertional inactivation of the LuxA and LuxB gene. This was done to see what effect this mutation had on the system overall. The two E. coli recombinants pJE201 and pJE202 were used as the main template for this step. Transposon Tn5 was used to mutate the recombinant plasmids through insertional inactivation. The figure below shows the various spots where the inserts were made and what was inserted. Determination of Bioluminescence: The final step was determine what effect, if any, these insertional inactivation mutations had on the regulatory system. Figure 5 shows the general layout of the genes and where they lie relative to each other. The researchers found that transposon insertion in operon R downstream from LuxA and LuxB did not effect luciferase synthesis. On the other hand, insertions upstream in operon R and operon L greatly reduced, but did not complete eliminate luciferase synthesis. Additionally, they also tested how the autoinducers were affected. The researchers found that these E. coli recombinants produce identical autoinducer molecules just like the isolated V. fischer''i strains.These results show that the way this system is regulated is by the use of autoinducers. References [http://biocuriousmembers.pbworks.com/w/file/fetch/50229051/EngebrechtCell83.pdf+ "Bacterial Bioluminescence: Isolation and Genetic Analysis of Functions from ''Vibrio fischeri"]'' ''Engebrecht, JoAnne et al, Cell, Vol. 32., 773-781, March 1983.